This proposal will determine whether the DNA repair protein AP endonuclease 2 (APE2) is important for the maintenance of hematopoietic stem cell (HSC) function and for the resistance of these cells to radiation. Very little is known about DNA repair mechanisms in the quiescent HSCs that retain multi-lineage repopulation potential throughout life, but protection of their genome is essential for the lifelong supply of blood cells. Genomic instability in HSCs is associated with aging phenotypes and leukemogenesis. Normally, HSC are relatively radioresistant, in part due to their quiescent state, which helps to protect them from DNA damage. However, radiation treatment, a widely used cancer therapy, generates tremendous amounts of reactive oxygen species that can cause breaks in DNA, even in non-dividing cells. APE2 is a recently identified repair protein, and very little is known about its in vivo function. I hypothesize that APE2 helps to repair single- strand DNA breaks in HSCs, and that this is important to protect the genomic integrity and function of HSCs from damage following radiation exposure. In addition, APE2 might also provide protection to HSC from endogenously acquired oxidative damage. This type of damage is associated with aging phenotypes in HSC and all cell types. In vitro, APE2 is efficient at removal of 3'-end blocking groups that prevent repair of DNA breaks, and many radiation-induced breaks have DNA ends that are blocked by 3'-phosphoglycolate moieties. I will test this hypothesis by exposing FACS-purified hematopoietic stem cells to increasing doses of ionizing radiation and assaying DNA damage, repair, and HSC function. The percent of cells that have single- strand and double-strand breaks will be determined by comet assay and by measuring 3H2AX foci, various times after irradiation. Untreated and irradiated HSC will be analyzed functionally in clonogenic survival assays using methylcellulose cultures and also by transplantation into irradiated recipient mice. I have already shown that APE2 is important for the rapid expansion of lymphoid precursors in the bone marrow during development, and that it is especially important during recovery of the bone marrow from chemotherapeutic depletion. The experiments proposed here will determine if APE2 is also important to protect the genomic stability of HSCs from endogenous genotoxic stress and from radiation exposure. Resistance of HSC to radiation is an important feature that allows recovery of the hematopoietic system in patients treated with radiation. However, the same factors that repair radiation damage in cells can also cause the resistance of HSC to treatments intended to eliminate cancer stem cells. Therefore, it is important to identify proteins that could cause resistance to treatment and that could be a target for combination therapies to increase efficacy. PUBLIC HEALTH RELEVANCE: Resistance of hematopoietic stem cells to radiation is an important feature that allows recovery of the hematopoietic system in patients treated with this widely used cancer therapy. These experiments are expected to identify the DNA repair protein AP endonuclease 2 (APE2) as a significant factor in stem cell resistance to radiation. Very little is known about the role of this enzyme in cells. Demonstration of a role for APE2 in the radioresistance of stem cells would also identify it as a potential target for combined therapies that would have increased effectiveness in the elimination of cancer stem cells prior to bone marrow transplantation.